Proteomics 2016, 16, 1331–1340 DOI 10.1002/pmic.

201500211 1331

RESEARCH ARTICLE

Dp71⌬78-79 dystrophin mutant stimulates neurite

outgrowth in PC12 cells via upregulation and
phosphorylation of HspB1
Candelaria Merino-Jiménez1 , Jorge Aragón1 , Vı́ctor Ceja1 , Griselda Rodrı́guez-Martı́nez1 ,
Febe E. Cázares-Raga2 , Solenne Chardonnet3,4 , Cédric Pionneau3,4 , Alvaro Rendon5
and Cecilia Montañez1
1
Departamento de Genética y Biologı́a Molecular, Centro de Investigación y de Estudios Avanzados del IPN,
México, D.F., México
2
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN,
México, D.F., México
3
UPMC Univ Paris 06, UMS 2 Omique, Sorbonne Universités, Plateforme P3S, Paris, France
4
UMS 29 Omique, INSERM, Plateforme P3S, Paris, France
5
Institut de la Vision, INSERM UMR_S968, CNRS UMR_7210, Université Pierre et Marie Curie Paris 06, Paris, France

PC12 cells acquire a neuronal phenotype in response to nerve growth factor (NGF). However, Received: June 4, 2015
this phenotype is more efficiently achieved when the Dp71⌬78-79 dystrophin mutant is stably Revised: January 24, 2016
expressed in PC12-C11 cells. To investigate the effect of Dp71⌬78-79 overexpression on the Accepted: February 29, 2016
protein profile of PC12-C11 cells, we compared the expression profiles of undifferentiated and
NGF-differentiated PC12-C11 and PC12 cells by 2DE. In undifferentiated cultures, one protein
was downregulated, and five were upregulated. Dp71⌬78-79 overexpression had a greater effect
on differentiated cultures, with ten proteins downregulated and seven upregulated. The protein
with the highest upregulation was HspB1. Changes in HspB1 expression were validated by
Western blot and immunofluorescence analyses. Interestingly, the neurite outgrowth in PC12-
C11 cells was affected by a polyclonal antibody against HspB1, and the level of HspB1 and
HspB1Ser86 decreased, suggesting an important role for this protein in this cellular process.
Our results show that Dp71⌬78-79 affects the expression level of some proteins and that the
stimulated neurite outgrowth produced by this mutant is mainly through upregulation and
phosphorylation of HspB1.

Keywords:
Cell biology / Dp71⌬78-79 / HspB1 / MS / Neurite outgrowth / PC12 cells

 Additional supporting information may be found in the online version of this article at
the publisher’s web-site

1 Introduction [1]. Dp71 transcripts are alternatively spliced in exons 71,

The dystrophin Dp71 protein is encoded by the Duchenne
muscular dystrophy (DMD) gene. It is expressed at high lev-
els in non-muscle tissues and is most abundant in adult brain
Correspondence: Dr. Cecilia Montañez, Departamento de
Genética y Biologı́a Molecular, Centro de Investigación y de Estu-
dios Avanzados del IPN, Av. IPN 2508, Col. San Pedro Zacatenco,
07360 México, D.F., México
E-mail: cecim@cinvestav.mx
Fax: +52-55-57473392
Abbreviations: NGF, neural growth factor; WB, Western blotting
71–74 and/or 78, generating several isoforms [2, 3]. Although
the function of Dp71 has not been fully elucidated, muta-
tions in the DMD gene that disrupt Dp71 expression have
been correlated with the severity of the cognitive impairment
observed in DMD patients, suggesting that Dp71 might play
an important role in the central nervous system [4–6].
Studies in the PC12 cell line have shown that the ex-
pression and localization of Dp71 isoforms are differentially
regulated during nerve growth factor (NGF)-induced
differentiation [7] and that the Dp71 isoform expression is
essential for neurite outgrowth [8]. Recently, our research


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1332 C. Merino-Jiménez et al.
Significance of the study
Our results show that the expression of the Dp71⌬78-79 dys-
trophin mutant modifies the expression profile of several
proteins. In addition, we demonstrate that the most upregu-
lated protein (HspB1) has an important role in neurite out-
group showed that a dystrophin Dp71⌬78-79 mutant lacking
exons 78 and 79 (PC12-C11 cells) stimulates NGF-induced
neuronal differentiation in PC12 cells [9]. In this study, we
investigated the effect of Dp71⌬78-79 overexpression on the
protein profiles of undifferentiated and NGF-differentiated
PC12-C11 cells using 2DE, with the aim of identifying differ-
entially regulated proteins controlled by Dp71⌬78-79 .
2 Materials and methods
2.1 Cell culture and differentiation
PC12-C11 and PC12 cells were grown and differentiated for
six days as previously described [9].
2.2 Protein sample preparation
Undifferentiated and differentiated PC12-C11 and PC12 cells
were harvested, washed with PBS and homogenized in a
dedicated 2DE lysis buffer (7 M urea, 2 M thiourea, 4%
CHAPS, 40 mM DTT, 2% IPG buffer, pH 3–10 NL) or in
a phosphorylation-preserving lysis buffer (20 mM Tris HCl,
pH 7.8, 1 mM EDTA, 10 mM ␤-glycerophosphate, 1 mM
Na3 CO4 , 11 mM NaF, 10 mM Na pyrophosphate) for specific
phosphoprotein detection. Both lysis buffers contained 1X of
protease inhibitor cocktail (Roche, Inc.). The proteins were
precipitated by the addition of ice-cold acetone, and the pellet
was dissolved in rehydration buffer (7 M urea, 2 M thiourea,
2% CHAPS, 40 mM DTT, 2% IPG buffer, pH 3–10 NL). Pro-
tein samples were cleaned using the 2-D Clean-Up kit (GE
Healthcare). The protein concentration was determined by
the Bradford method.
2.3 2DE
For the IEF, IPG strips (pH 3.0–10.0 NL, 7 cm) were rehy-
drated for 16 h with rehydration buffer containing 200 ␮g
of protein and 0.05% bromophenol. The strips were run on
an Ettan IPGphor 3 Isoelectric Focusing System (GE Health-
care), with the following running conditions: step-and-hold at
300 V (200 Vh), gradient to 1000 V (300 Vh), gradient to 5000 V
(4500 Vh) and finally step-and-hold at 5000 V (2000 Vh).
After IEF, strips were immersed in equilibration buffer (6 M

C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2016, 16, 1331–1340
growth of PC12-C11 cells. These results provide the basis to
explore the mechanism by which the Dp71 mutant promotes
neurite outgrowth via enhanced expression and phosphory-
lation of HspB1 in PC12 cells.
urea, 30% v/v glycerol, 2% w/v SDS, 75 mM Tris-HCl, pH
8.8, 0.002% w/v bromophenol blue and 1% w/v DTT) for 15
min, followed by a second treatment in the same solution
where the DTT was replaced by 2.5% w/v iodoacetamide.
Then, equilibrated strips were run in 12% SDS-PAGE using
a Mini-Protean Tetra system (Bio-Rad) at 80 V/gel for 3 h.
Gels were stained with Bio-Safe Coomassie Stain (Bio-Rad).
Four 2DE gels were obtained using independent biological
replicates for each condition.
2.4 Image and data analysis
2DE gels were scanned with the Gel DocTM EZ Imager (Bio-
Rad). To evaluate protein abundance, digitalized gel images
were analyzed using ImageMaster 2D Platinum 6.0 software
(GE Healthcare). The percentage of volume (% volume) was
determined for each spot. Resulting sets of averaged rela-
tive spot volume were analyzed by a non-parametric Mann–
Whitney test to detect spots with different expression levels.
Spots with P-values<0.05 were considered statistically signif-
icant. The spots with changes of 1.5-fold or more were excised
from the 2DE gels and analyzed by MS.
2.5 Protein identification
Excised spots were destained in 50% v/v ethanol, 50 mM
NH4 HCO3 and dehydrated with ACN. Gel pieces were di-
gested overnight with 200 ng of trypsin (G-Biosciences) in
50 mM NH4 HCO3 , 5% ACN. Supernatants were collected,
and gel pieces were washed twice with 50% ACN, 0.1% TFA
in an ultrasonic bath. Peptides were vacuum dried and re-
suspended in 30% ACN, 0.1% TFA. For MALDI-TOF MS
analysis, 1 ␮L of peptide solution was mixed with 1 ␮L of ma-
trix solution (0.7 mg/mL CHCA in 50% ACN, 0.1% TFA) and
analyzed using an Autoflex Speed MALDI-TOF/TOF mass
spectrometer (Bruker Daltonics) in reflectron mode or using
a 4800 Plus MALDI TOF/TOF mass spectrometer (Sciex). LC-
MS/MS analysis was performed with an Ultimate3000 Nano
HPLC (Thermo Fisher Scientific) coupled with an HCT ultra
ion trap mass spectrometer (Bruker Daltonics). LC separation
was performed on a PepMap100 column (0.075 mm id, 3-␮m
particle size, 15 cm) at a flow rate of 300 nL/min with a linear
gradient from 0 to 35% of H2 O/ACN/formic acid (5/95/0.1
v/v) for 35 min. Proteins were identified using MASCOT
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Proteomics 2016, 16, 1331–1340
version 2.5.1 (MatrixScience) against the Swiss-Prot, TrEMBL
or NCBInr of the Rattus norvegicus data bank, allowing for
two missed cleavages with carbamidomethylation of cysteines
(fixed modification), oxidation of methionine (variable modi-
fication), and MS and MS/MS tolerance of 0.5 Da. Confident
matches were defined by a MASCOT score higher than 52 for
MALDI-TOF and at least two peptides with a score of 31 for
LC-MS/MS (p-value<0.05).
2.6 Western blot analysis
Forty ␮g of total proteins was used for Western blot (WB)
assays as previously described [9]. Rabbit polyclonal an-
tibodies against HspB1, annexin-5, TH, aldose reductase,
phospho-specific HspB1Ser86 and mouse monoclonal an-
tibodies against secretogranin-2 and NF-L were purchased
from Abcam. The mouse monoclonal anti-␤-actin was pro-
vided by Dr. Manuel Hernández (CINVESTAV, México). Im-
munoreactivity was detected by the Lightning Plus ECL kit
(PerkinElmer, Inc.). Images were captured using the Kodak
Digital Science ID programme. The results of densitometry
analyses of WB, obtained using ImageJ software (US National
Institutes of Health), were represented as relative density to
the control (␤-actin). Assays were performed in triplicate.
2.7 Immunofluorescence assays
Differentiated PC12-C11 and PC12 cells were grown on cov-
erslips precoated with poly-L-lysine and fixed with methanol.
Cells were permeabilized with 0.3% Triton X-100 and blocked
with 0.5% gelatin. Primary antibodies (rabbit polyclonal
anti-HspB1, anti-annexin-5 (Abcam) and mouse monoclonal
anti-SYP (Santa Cruz)) were incubated overnight at 4⬚C.
Immunolabeling was visualized with Alexa-488- or Alexa-
594-conjugated secondary antibodies (Molecular Probes, In-
vitrogen). Nuclei were counterstained with DAPI (1 ng/mL,
Sigma). Mounting was performed in Vectashield mounting
medium (Vector Laboratories, Inc.). Immunofluorescence
was visualized by confocal microscopy (Leica TCS SP8). 15–20
optical Z-sections (0.3–0.5 ␮m thick) were scanned from each
sample. To quantify the fluorescence intensity of each pro-
tein, thirty photomicrographs of 1014×1014 pixels acquired
at 40X magnification with the Leica TCS SP8 were analyzed
using the Leica TCS SP software.
2.8 Immunologic blockade assay
PC12-C11 and PC12 cells were grown to 70% confluence on
collagen-coated culture dishes and treated with 50 ng/mL
NGF (Invitrogen) for 3 days in presence of 10 ␮M of
anti-HspB1 antibody (sc-1049, Santa Cruz) or the vehicle.
Morphological changes were observed using an Axiovert25
Zeiss microscope at 40X magnification. For each treatment

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1333
group, ten photomicrographs were analyzed, and the percent-
age of cells bearing neurites at least one cell body length was
enumerated using the ImageJ software (US National Insti-
tutes of Health). Assays were performed in triplicate. The
expression levels of HspB1 and HspB1Ser86 were measured
by WB after treatment. Assays were performed in duplicate.
2.9 Statistical analysis
The results obtained from three independent experiments
are expressed as the mean ± SD. Statistical analysis was
performed by an unpaired Student’s t-test using GraphPad
Prism 5 software. P-values<0.05 were considered statistically
significant for the quantification of the relative expression of
the selected proteins.
3 Results
3.1 Protein expression profiles of undifferentiated
and differentiated PC12-C11 cells
To better understand the role of the Dp71⌬78-79 dystrophin
mutant in the stimulation of PC12-C11 neuronal differenti-
ation [9], we compared the expression profiles of undiffer-
entiated and NGF-differentiated PC12-C11 and PC12 cells
(Fig. 1). Proteins were considered as differentially expressed
with at least a 1.5-fold change. In undifferentiated PC12-C11
cells, 323 protein spots were detected, but only four spots (U1-
U4, where U represents undifferentiated cells) showed differ-
ential expression (Fig. 1A and B). In differentiated PC12-C11
cells, 391 protein spots were detected, and only 15 spots (D1-
D15, D for differentiated cells) showed differential expression
(Fig. 1C and D). Magnified views of all differentially expressed
proteins in the presence of NGF are presented in Fig. 1E. The
% volume for each differentially expressed spot is shown in
Fig. 1F. Overall, these results indicate that some proteins
are differentially expressed due to the Dp71⌬78-79 dystrophin
mutant expression in PC12 cells.
3.2 Protein identification
To identify the differentially expressed proteins, the selected
spots were excised from 2DE gels and analyzed by MS. A
total of 23 proteins were identified (Table 1), and in some
spots, more than two proteins were found. We selected the
proteins with higher numbers of matched peptides. In some
cases, the same protein was identified in two different spots,
corresponding to distinct isoforms of the protein, which was
the case for annexin-2, GAPDH and HspB1. Among the 17
proteins differentially expressed in differentiated cells, the
most substantially upregulated protein was HspB1, with two
different isoforms that were 5.2-fold (D13) and 3.5-fold (D14)
overexpressed in PC12-C11 cells (Table 1).
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1334 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

Figure 1. Representative 2DE maps of PC12-C11 and PC12 proteins. (A and B) Protein profiles of undifferentiated (U) PC12-C11 and PC12
cells, respectively. U1-U4 indicates differentially expressed proteins. (C and D) Protein profiles of differentiated (D) PC12-C11 and PC12 cells,
respectively. D1-D15 indicates differentially expressed proteins. (E) Magnified views of the PC12-C11 and PC12 spots that were differentially
expressed in NGF-differentiated cells. (F) The % Volume of spots differentially expressed in PC12-C11 and PC12 NGF-differentiated cells.
The graph shows the average % Volume ± SD for four separate experiments. ** P<0.01 and *** P<0.001. The arrow represents the
nonlinear immobilized pH gradient used for IEF. Positions of standard mass markers (kDa) for the second dimension are shown on the left
side of the images.


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 Table 1. Proteins identified by mass spectrometry


Proteomics 2016, 16, 1331–1340

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Spot ID Variation Fold-change MS Accession Protein Gene MW [kDa] pI MASCOT Match SC (%)b)
in spot no.a) score Pept

U1 – 2.5 LC-MS/MS Q62952 Dihydropyrimidinase-related Dpysl3 61.9 6.0 1359.5 24 45.3

protein 3
U2 + 3.7 LC-MS/MS P85834 Elongation factor Tu, Tufm 49.5 7.9 1321.0 23 60.8
mitochondrial
P41562 Isocitrate dehydrogenase Idh1 46.7 6.6 835.6 16 37.9
[NADP] cytoplasmic
U3 + 2.3 LC-MS/MS O88767 Protein DJ-1 Park7 20.0 6.4 506.8 10 56.6
U4 + n.a. LC-MS/MS P62260 14-3-3 protein epsilon Ywhae 29.2 4.5 1096.1 19 79.6
Q63610 Tropomyosin alpha-3 chain Tpm3 29.0 4.6 699.1 11 40.3
D1 + 2.3 LC-MS/MS Q66HD0 Endoplasmin Hsp90b1 92.7 4.6 1923.8 33 37.1
D2 – 6.1 LC-MS/MS P10362 Secretogranin-2 Scg2 71.0 4.6 1348.9 28 46.4
P20156 Neurosecretory protein VGF Vgf 68.1 4.5 1037.2 16 33.4
D3 + 1.7 LC-MS/MS P06761 78 kDa glucose-regulated Hspa5 72.3 4.9 2563.2 38 48.8
protein
D4 – n.a. LC-MS/MS P19527 Neurofilament light Nefl 61.3 4.5 1648.5 30 47.2
polypeptide
D5 – 2.9 LC-MS/MS Q62952 Dihydropyrimidinase-related Dpysl3 61.9 6.0 1542.9 28 54
protein 3
P04177 Tyrosine 3-monooxygenase Th 55.9 5.7 575.5 9 33.1
D6 + 1.9 LC-MS/MS P18418 Calreticulin Calr 48.0 4.2 679.9 14 30
D7 – 2.2 LC-MS/MS B1WC26 N-acetylneuraminic acid Nans 40.0 6.4 633.1 13 30.9
synthase
Q5XI22 Acetyl-CoA acetyltransferase, Acat2 41.1 7.1 513.1 8 37.3
cytosolic
D8 – 3.4 LC-MS/MS Q5XI22 Acetyl-CoA acetyltransferase, Acat2 41.1 7.1 732.1 10 65
cytosolic
Q64559 Cytosolic acyl coenzyme A Acot7 42.7 9.6 424.3 7 21.5
thioester hydrolase
D9 – 1.6 MALDI P07943 Aldose reductase Akr1b1 35.8 6.3 124.0 13 46.8
D10 + 1.9 LC-MS/MS Q07936 Annexin A2 Anxa2 38.7 8.6 1587.6 25 62.8
P04797 Glyceraldehyde-3-phosphate Gapdh 35.8 9.0 701.2 14 41.7
dehydrogenase
D11 + 2.1 LC-MS/MS Q07936 Annexin A2 Anxa2 38.7 8.6 1507.8 25 64
P04797 Glyceraldehyde-3-phosphate Gapdh 35.8 9.0 561.4 11 28.2
dehydrogenase
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D12 + 2.2 MALDI P14668 Annexin A5 Anxa5 35.7 4.9 926.0 15 66.5
D13 + 3.5 MALDI P42930 Heat shock protein beta-1 Hspb1 22.9 6.1 161.0 13 63.1
D14 + 5.2 MALDI P42930 Heat shock protein beta-1 Hspb1 22.9 6.1 690.0 12 82
D15 – n.a. LC-MS/MS Q00981 Ubiquitin carboxyl-terminal Uchl1 24.8 5.0 541.1 10 53.8
hydrolase isozyme L1
Q5XI73 Rho GDP-dissociation inhibitor Arhgdia 23.4 5.0 448.1 9 30.4
1
a) Accession number according to the Swiss-Prot database of Rattus norvegicus.

1335
b) SC % = % of the sequence identified.
n.a.: not applicable as % volume was only obtained for one of the two PC12-C11 or PC12 cells.
1336 C. Merino-Jiménez et al.
3.3 Validation of differentially expressed proteins
To validate the changes in the expression levels observed in
the 2DE gels, we focused on HspB1 and annexin-5 because
the first one is implicated in neurite outgrowth [10], and
annexin-5 is a membrane-associated protein with calcium-
channel activity [11]. In addition, we also selected some pro-
teins involved in PC12 differentiation induced by NGF that
unexpectedly were downregulated by Dp71⌬78-79 dystrophin
expression (NF-L, secretogranin-2, TH and aldose reductase).
For all the selected proteins, the relative expression levels
were measured in differentiated PC12-C11 and PC12 cells
by WB (Fig. 2). Similarly to the results obtained by 2DE
(Fig. 1), HspB1 exhibited a 6.3-fold increase, and annexin-
5 showed a 2.1-fold increase, while NF-L decreased 8.4-fold,
secretogranin-2 5.1-fold, TH 2.3-fold and aldose reductase
2.4-fold (Fig. 2).
3.4 Localization of HspB1 and annexin-5 proteins
To determine the localization of HspB1 and annexin-5, an
immunofluorescence assay was performed in differentiated
PC12-C11 and PC12 cells using the rabbit polyclonal anti-
HspB1 and anti-annexin-5 antibodies. In both panels of
Fig. 3, the neuronal marker synaptophysin is shown in green,
HspB1 or annexin-5 are shown in red, and nuclei stained with
DAPI are represented in blue. The localization of HspB1
in differentiated PC12-C11 cells was mainly cytoplasmic, al-
though it was also observed along the neurites (Fig. 3B). In
contrast, in PC12 cells, immunostaining of HspB1 was barely
observed (Fig. 3E). Annexin-5 was predominantly cytoplasmic
and nuclear and was observed along the length of the neu-
rites in PC12-C11 cells (Fig. 3H), but in PC12 cells, it was
cytoplasmic, although the immunostaining was lower than
in PC12-C11 cells (Fig. 3K). The fluorescence intensity quan-

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Proteomics 2016, 16, 1331–1340
Figure 2. Validation of differen-
tially expressed proteins. (A) Ex-
pression of HspB1, annexin-5,
NF-L, secretogranin-2, TH and
aldose reductase proteins. (B)
Relative expression of HspB1,
annexin-5, NF-L, secretogranin-
2, TH and aldose reductase pro-
teins, respectively, normalized
to ␤-actin expression. The graph
presents the mean ± SD from
three independent experiments.
* P < 0.05, ** P < 0.01 and ***
P < 0.001.
tification showed that HspB1 immunoreactivity was 3.3-fold
higher in PC12-C11 compared to PC12 cells, whereas for
annexin-5, it was 2.0-fold higher (Fig. 3M).
3.5 Immunologic blockade assay against HspB1
To study the role of HspB1 during NGF-induced differen-
tiation of PC12-C11 cells, we employed an immunologic
blockade assay using an antibody against HspB1. The cell
differentiation percentage was monitored on the third day
of treatment. PC12-C11 cells treated with NGF showed
20.71% ± 0.97 differentiation and NGF + control (vehicle
of polyclonal anti-HspB1) showed 21.70% ± 1.36, but in
the presence of NGF + anti-HspB1, the percentage of cell
differentiation decreased considerably to 5.38% ± 1.36. In
PC12 cells treated with NGF, 15.94% ± 1.44 of cell differen-
tiation was observed, NFG + control was 15.59% ± 0.86, and
NGF + anti-HspB1 was 12.26% ± 1.85. Therefore, our results
show that treatment with anti-HspB1 decreased the number
of neurite-bearing cells (Fig. 4C and G). It has been shown
that HspB1 is activated upon phosphorylation [10, 12–14].
Ser15, Ser78 and Ser82 are the main sites for phosphoryla-
tion in human, and Ser15 and Ser86 are the main sites in rat
(Ser82 is the major phosphorylation site and an orthologous
site of HspB1Ser86) [15, 16]. To determine the effect of the
immunologic blockade assay, we measured the expression
levels of HspB1 and HspB1Ser86 by WB. Interestingly, both
HspB1 and HspB1Ser86 decreased after the treatment with
the antibody (Fig. 4H and I).
4 Discussion
In this study, to elucidate how Dp71⌬78-79 stimulates neu-
ronal differentiation, we compared the proteomic expression
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Proteomics 2016, 16, 1331–1340 1337

Figure 3. Localization of HspB1

and annexin-5 in PC12-C11 and
PC12 differentiated cells. A, D,
G and J depict the localiza-
tion of the synaptophysin pro-
tein (in green) used as a neu-
ronal marker. (B and E) Localiza-
tion of HspB1 (red) in PC12-C11
and PC12 cells, respectively. (H
and K) Localization of annexin-
5 (red) in PC12-C11 and PC12
cells, respectively. C, F, I and
L show the merge of the two
preceding images. Nuclei were
stained with DAPI (blue). Images
are representative of three inde-
pendent experiments. Scale bar
represents 25 ␮m. (M) Quantita-
tion of HspB1 and annexin-5 flu-
orescence intensity. The graph
represents the mean ± SD from
three independent experiments.
** P < 0.01 and *** P < 0.001.


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1338 C. Merino-Jiménez et al. Proteomics 2016, 16, 1331–1340

Figure 4. Anti-HspB1 effect upon neurite outgrowth in PC12-C11 cells. (A–C) PC12-C11 cells, (D–F) PC12 cells. (A and D) Cells treated with
NGF. (B and E) NGF + control (vehicle of polyclonal anti-HspB1), (C and F) NGF + anti-HspB1. (G) Percentage of cells with neurites on
the third day of treatment. The graph represents the mean ± SD of three independent experiments. The scale bar represents 50 ␮m.
(H) Detection of HspB1 and HspBSer86 in cells treated with anti-HspB1. (I) Relative expression of HspB1 normalized to ␤-actin expression
and relative expression of HspB1Ser86 normalized to HspB1 expression. The graph represents the mean ± SD from two independent
experiments. * P < 0.05 and *** P < 0.001.

between PC12-C11 and PC12 cells. We identified several pro-
teins in most of the variant spots because the use of 7 cm 2DE
gels resulted in a co-migration in the same spot of proteins
having a close pI and molecular weight. We thus considered
that proteins with at least twice as many peptides as others
were responsible for the variation observed; however, we can-
not exclude an effect of some of the less abundant proteins
that were observed (Supporting Information Table 1).
In undifferentiated cultures, DRP-3 was identified as a
downregulated protein, which has been associated with class
3 semaphorin signaling, axon guidance [17], neuronal growth
cone collapse and cell migration. Interestingly, the expres-
sion levels of this protein also decreased in differentiated
cultures, making it important to explore its effect on the
growth cone in PC12-C11 differentiated cells. Among the
upregulated proteins identified, the 14-3-3E protein has been
reported to enhance catecholamine exocytosis [18] by disas-
sembling the actin network to facilitate the movement of
secretory granules [19]. Tropomyosin-3 is implicated in the
stabilization of cytoskeleton actin filaments. The increased
expression of both proteins could explain the morphological
changes observed in undifferentiated PC12-C11 cells; PC12
cells are round, whereas the PC12-C11 cells are oval and even
begin to show neurite outgrowth [9]. DJ-1 has a protective role
against oxidative stress [20]. Therefore, the observed increase
of this protein may suggest a neuro-protective effect.
The effect of Dp71⌬78-79 on the protein profile of NGF-
differentiated PC12-C11 cells was more evident. Annexin-
5 and annexin-2 were upregulated proteins. Annexins are
calcium-binding proteins involved in membrane traffick-
ing, membrane-cytoskeleton interactions, calcium channel
activity and signal transduction [21]. Annexin A2 bundles
actin filaments and plays a role in their polymerization
[22], whereas annexin A5 modulates the affinity for cal-
cium ions to create cooperative binding [23]. The increased
expression of these proteins may suggest a role as ion
channels to regulate cytosolic calcium levels in PC12-C11
cells.
HspB1 showed the highest increase, and it has been de-
scribed in filament structures within dendrites or axons and


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Proteomics 2016, 16, 1331–1340
in synaptic components [14]. Phosphorylated HspB1 is in-
volved in cytoskeleton restructuration, actin polymerization
and stress fibre formation, contributing to axonal outgrowth
[10, 12, 13]. In PC12-C11 cells, the HspB1 protein was located
in the cytoplasm and along the neurites (Fig. 3B). In addition,
we determined the biological relevance of HspB1 with an
immunologic blockade assay using a polyclonal anti-HspB1.
In this experiment, the neurite outgrowth of PC12-C11 cells
was significantly reduced as well as the level of HspB1 and
HspB1Ser86, suggesting that the expression and phosphory-
lation of HspB1 are required for neurite outgrowth of PC12-
C11 cells. On the other hand, the neurite outgrowth of NGF-
differentiated PC12 cells was not considerable affected by the
treatment with anti-HspB1 because the HspB1 expression in
these cells is very low. These results suggest that the neu-
rite outgrowth in PC12 cells is independent of HspB1 in the
presence of NGF. However, it has been reported that when
PC12 cells are stimulated to induce neurite outgrowth by
pituitary adenylate cyclase-activating polypeptide (PACAP),
HspB1 was upregulated [24]. Furthermore, in a subline of
PC12 cells (PC6), the neurite outgrowth was dependent on
HspB1Ser82 activation through PKA [25].
In this work, we found that differentiated PC12-C11 cells
show phosphorylation of HspB1Ser86, suggesting an activa-
tion of this protein, which could be responsible for promoting
neurite outgrowth. Based on all results obtained, HspB1 is a
likely to be a relevant protein for neurite outgrowth in PC12-
C11 cells.
Surprisingly, some proteins that play important roles
in PC12 neuronal differentiation were downregulated
(secretogranin-2, TH, aldose reductase and NF-L) (Fig. 2).
Secretogranin-2 is involved in secretion and increases during
NGF treatment [26]. TH catalyses the conversion of tyrosine
to L-DOPA, the first step in biosynthesis of catecholamines
[27]. Aldose reductase participates in the reduction of 3,4-
dihydroxyphenylacetaldehyde (DOPAL), a toxic metabolite
formed by the oxidative deamination of dopamine to 3,4-
dihydroxyphenylethanol (DOPET) [28]. The downregulation
of these proteins in PC12-C11 cells may suggest an alteration
in the secretion of catecholamines, mainly dopamine. Neuro-
filament proteins contribute to the extension and stabilization
of neuronal processes, and it has been demonstrated that the
degree of PC12 differentiation can be quantified by NF-L ex-
pression [29]. Rho GDI 1 is also downregulated in PC12-C11
cells and is implicated in the reorganization of the actin cy-
toskeleton [30]. The downregulation of proteins relevant to
neuronal differentiation suggests that the observed neurite
outgrowth requires lower levels of these proteins compared
to the PC12 cells.
Annexins, tropomyosins and heat shock proteins are com-
monly found as variant proteins in proteomic studies and are
not considered to be relevant proteins [31]. It is not the case for
this study, where the changes in the expression of annexin-5
and HspB1 were validated by WB and immunofluorescence
assays. Moreover, the biological relevance of the upregulated
HspB1 in neurite outgrowth was also demonstrated.

C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1339
Based on the findings described in this work, our results
showed that the expression of the Dp71⌬78-79 dystrophin mu-
tant altered the protein expression profile of PC12 cells and
that the induced neuritogenesis takes place via upregulation
and phosphorylation of HspB1.
We gratefully acknowledge Consejo Nacional de Ciencia y Tec-
nologı́a (Conacyt) for supporting this work (C. Merino Doctoral
Fellowship 233893 and C. Montañez Grant CB-2013-222054
and ECOS-ANUIES-SEP-Conacyt M11-S02). We thank MSc.
Emmanuel Rı́os Castro at the Unidad de Genómica, Protéomica
y Metabolómica, LaNSE-CINVESTAV for equipment support.
The authors have no conflicts of interest to declare.
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